U N IT E D N A T IO N S E N V IR O N M E N T P R O G R A M M E
O C T O B E R 1995
D e te rm in a tio n o f
fa e c a l c o lifo rm s in
sea w ater by the m em brane
f ilt r a tio n (M F ) cu ltu re m ethod
Reference M ethods F o r M a rin e P o llu tio n Studies N o. 3 (Rev. I )
P re p a re d in c o -o p e ra tio u w ilh
W HO
UNEP
1995
T h is dosu irie u t lu s been prepared b> th e W ot Id H e a ld i O riy u n Tattoo (W H U ) a n d issued ov the
Interna i icna I A to m ic Cncrgv Agency. M a rin o lim jr o iu u c n t b ib o rm m y ( tA J 'A -M E L i a n d ih c United
N ations b n v iro n m e n t P to g n rn m e ( V N t P i u n der project F P /M F /s i u I -<) 1-1)30033).
F o r t>il)!iographic purposes th is docum ent m a yb e cite d as:
T.iNF.P/W liO .
D e te rm in a tio n o f fa c u il c o ltfo m is in sea w a te r b j ltu¿ m em brane filtra Q o n ( M i') culture
m ethod
Reference M ethods f o r M a m ie P o llu tio n Studies N o 3. R ev 1 U N F P , '995
PREFACE
T h e R egional Seas Program m e was in itia te d b y U N E P in 1974
Since chen the (îo v c riu n g
C o u n c il o f U N E P has le p c a lu ilv endorsed a re g io n a l approach to Ih c c o n tro l o f m ariae p o ll in io n and }he
m anagem ent o f m a rin e a n d coastal resources, a n d bas requeued the developm ent o f re g io n a l action plans
I'he R e g io nar Seas Program m e nt present includes 12 regions a n d has over Í4 0 coascaJ stales p a rtic ip a tin g
in it ( I ) , (2).
O n e o f die basic components o f th e a c tio n pla n s sponsored by U N E P in the fra m e w o rk o f the
R egional Seas P rogram m e ;s d ic assessment o f d ie state o f the m arine e n viro n m e n t and < S n«; rewurces.
and o f die sources and trends o f the p o llu tio n and the im p a c t o f p o llu tio n o n hum an health, m ín n e
ccusystems a n d am enities In o rd e r to assist those p a rtic ip a tin g in th is a c tiv ity , a n d to ensure th n f the data
o btajjie d tiu o u g k th is asscsMiicnt c«ui be compared o n a w o rld -w id e basts a n d thus co ntribute to the Global
E n viro n m e n t M o n ito rin g System (G E M S ) o f U NEP, a sot o f Rcfereoce M ethods a n d G in del ¡nes fo r
m arine p o llu tio n studies h being developed as p a r: o f a jtfo g ra m n ic o f com prehensive te ch n ica l support
w h ic h includes liae p ro v is m a o f expert advice, reference methods and iru ite n a ls, tra in in g a n d d n u i (p c iliiy
assurance ( : ) T h e methods *re recommended to be adopted b y Governm ents p a rtic ip a tin g in the Regional
Sens Program m e
T h e m edlinia snd guidelines are prepajed b y , o r m cooperation w ith , the relevant speeui)i7ed
I h k I ic s o f d ic U n ite d N a tio n s system as w e ll a s o ilie r orga n iza tio n s. a n d are tested by a num ber o f expert«
com petent in the fie ld relevant to the methods described
I i i the de scrip tio n o f the methods and guidelines the stvle used by th e Inte rn a tio n a l O r g ;in i7 * t to n
fo r S bu idarduaU on (ISO) rs fo llo w e d as c lo s e ly as possible
T h e m e tlio d s and guideline*, as published i n U NEP's series o f R eference M ethods fo r M a rin e
P o llu tio n Studies, « u t n o t considered us fin a l. T h e y are p ia o ne d to b e p e rio d ic a lly revised ta k in g in to
account the developm ent o i o n i understanding o f th e problem s, o f a n a lytica l in s tru m e n ta tio n and the
actual need o f the users. In order to fa c ilita te these revisions, the users are in v ite d to convey th e ir
com m ents a n d suggestions lo*
W H O /E U R O Project O ffice
C o o rd in a tin g U n it fo r the M editerranean A c non Plan
48 Vussileos K o nstantinou
P.O. B o x 1801V
G R -i 1610 Athens
GREECE
w h ic h is responsible fo r the developm ent a n d preparation o f m ic ro b io lo g ic a l and o ilie r heaJQi-rclated
Reference M ethods.
( l i liN F ,P .
A d ile V«cuen Is ojkJ p la n n e d d s v e lc p m e « '. o f U i i 1,‘ N h F s R e g io n a l S e r^ P m ^ n r m i * » i d
i. iin i(ia i j | i ) e p c ^ ^ o i/ a u « v p o n s u m l b y o ü ie r b o l l e *
C N Ü P R c r i a iá J S e i« fîc p o r ts a n d
«¡iiiitiwMft l. L'riLf'. h>82
<2JK IIULM-
A s ifM îg > t e r 'lio Seas. T lic R egional S e is P eoftrajionc. P a *l and fu t u r s , U N E P l f ? 0 .
G ) t." N E P /lA fc A 'lU C
R clè isn cs M e th o d* . m l M -d s n a !* v f*r<'gr.u*ime f i r cionprshesistvc s u p p o rt l o r re g io n a l
and g lo b a l m u jiu e p o lJ u ú c n u J s e ftm e o tí U N L P . 1SSHJ.
T in « revised issue o f Reference M ethods fo r M a rin e P o llu tio n Studies N o. 3 was prepared by the
W o rld H e a lth O rga n iza tio n o n the basis o f a review o f the M e th o d d u rin g e xp e rt m eetings and com m ents
fro m in d iv id u a l scientists w ho tested th e M e th o d T h e assistance o f a ll (hose w ho contrib u ted to th is
revised issue o f the Reference M e th o d is g ra te fu lly aclaioivledged
—i i i —
CO NTENTS
Page
1.
In tro d u ctio n
1
2.
S co p e a nd field c i a pplication
1
3.
D efinition
1
4.
Principles
2
5.
A pp a ra tu s a nd glassw are
2
S.
C u ltu re m edia, cn em icais and s to c k cu lture
5
7.
S am pling
7
8.
Test p ro ce d u re
8
9.
E xpression o f results
12
to .
Test re p o rt
14
11,
References
16
1
1.
IN T R O D U C T IO N
The o rig in al versio n o f this re co m m e n d e d m e th o d w a s prepared b y th e W orld
Health O rganization w ithin the fra m e w o rk o f th e L on g -term P ro gram m e o f Pollution
M onitoring a nd R esearch in th e M editerranean S ea (M ED P O L Phase II) a nd issued by
th e U nited N ations E nvironm ent P ro gram m e as R eference M e th o d fo r M arine P ollution
S tud ie s N o. 3 w ithin UNEP’s Regional Seas P ro gram m e A ctivity C entre’s series.
The m e th o d is essentially b a se d o n a lready-existing re co gn ize d tech niques, and
also d ra w n o n th e experience o f m icro b io lo g ists in a n u m b e r o f M editerranean
laboratories. In th e d e scrip tio n , th e style u se d b y th e International O rganization fo r
S tandardization (ISO) is follo w ed a s clo se ly a s p o ssib le . W hile d esign e d prim arily w ith
co n d itio n s pre vailin g w ithin th e M editerranean S e a in m in d , th e m e th o d ts a lso , to
variable e xtents, su ita b le fo r o th e r sim ila r e co lo g ica l re g ion s.
The p re sen t versio n o f this m e th o d in co rp o ra te s a n u m b e r o f a m e ndm ents,
based o n reviews d u rin g expert co nsu lta tion m e e tin g s o rg an ize d b y W H O , a nd o n
c o m m e n ts received fro m M editerranean la b ora to rie s u sin g th e m e tho d w ith in th e
fra m e w o rk o f th e ir n ational o r tocaJ m arine p ollu tio n m o n ito rin g program m e.
2.
S C O P E A N D F IE L D O F A P P L IC A T IO N
The m e th o d d e scribe d is suitable for the d ete rm ina tio n o f faecal co lifo rm s in
coastal b ath in g w aters o f tem perate a nd tro pica! seas. It is designed to be used in
sa nitary surveillance o í b ath in g beaches.
it uses a m e m b ra n e filter p rocedure w hich a llo w s concentration o f th e bacteria
p rie r to in cu ba tio n,
it can be e m p lo ye d in alternation w ith th e M ultiple*Tube
F erm entation (MPN) T e st (U N E P /W H O 1983). W h e th e r th e M e m brane Filtration (MF)
C ulture M e tho d is preferred to the MPN T e st d epends o n local c o n d itio n s a n d personal
preferences, in general, th e M r m e tho d is less la b or-in te n sive and, due to the
p re con ce ntra tio n o f th e b acte ria in th e sa m ple, it is m o re suitable in situabons w here low
num be rs o f co lifo rm s are to be estim ated. The M PN te s t s h o u ld b e given preference
when th e te s t sam p le co n ta in s high a m ounts o f particula te m a tte r w hich will hinder the
reading o f th e M Fs a fte r incubation.
Faecal co lifo rm s e xhibit a nighJy sp ecific p ositive correlation w ith faecal
co nta m in a tion fro m w a rm -b lo o d e d anim als, a nd the re fo re are g o o d in d icato rs fo r th e
sa nitary q ua lity o f coastal w aters. S ince fae ca l co lifo rm s d ie w ithin h o u rs w h e n exposed
to s u n lig h t In seaw ater a t tem pe ra tures a b o ve + 4 0 C, th e ir presence in seaw ater
indicates o n ly recent co n ta m in a tio n oy faecal m aterial. Die-away rates (T-90) depend
on salinity, tem perature, solar radiation, e tc a nd m u s t be taken into co nside ra tion when
interpreting th e results.
3.
D E F IN IT IO N
Faecal co n fo rm s ace a e ro b ic a nd fa cu lta tive ly a n a e ro b ic, G ram -negative, non*
s p o re fo rm in g ro d s tha t fe rm e n t la cto se w n ile p ro d u cin g a cid a n d gas, b oth a t 36° C and
44.5° C , in less tha n 2 4 hours. T hey p ro d u ce in d o le in tryp to n e w ater co nta in in g
2
tryp to p h a n a t 4 4.5 ° C. U nder th e co n d itio n s d e scrib e d in th is reference m e th o d , the
faecal co lifo rm s a p p e a r as blue colonies.
4.
P R IN C IP L E S
From sea-w ater sa m p le s ta ke n u n d e r stehle co n d itio n s, a d ilution series is set
u p a c c o rd in g to th e n u m b e r o t tae ca i c o lifo rm s e xp e c te d in th e w a te r sa m p le . A liq uo ts
o f this d ilu tio n se rie s arc filte re d th ro u g h 0 .4 5 y p ore size m e m b ra n e filters. The
m e m b ra n e filters are pla ced o n th e surface o f m *FC a ga r c o n ta in e d In Petri dish es and
in cu b a te d a t 4 4 .5 : 0 .2 ° C fo r 2 4 h o u rs. Lactose fe rm e n ta tio n will ca use co lo n ie s o f
faecal co lifo rm s (3) to e xhibit a ch a ra cte ristic b lu e c o lo u r.
R esidual chlorine, if present, is n eu tra lize d b y a d d in g th io su lp h a te to d ie
s a m p lin g b o ttle b e fo re sterilization.
S u sp e ct a n d d ou b tfu l co lo n ie s can be te ste d fo r a cid a n d gas d e ve lo p m e n t with
a co nfirm a tive te s t u sin g M scC o n ke y b ro th o r b rillia n t g reen bUe b ro th . In areas w here
in d ustrie s d isch a rg e polysaccharites (p a p e r m ills, s u g a r b ee t industries, etc.), furthe r
co n firm a tio n b y th e in d ole te s t m ay b e necessary.
5.
APPARATU S AND GLASSW ARE
5.1
S am p le b attle s o f b orosilica le g la s s fo r surface seaw ater, 200*300 m l capacity,
w id e -m o u th e d a nd w ith g ro u n d -g la ss stoppers.
5 .2
S am p le ro d o f n on -corrosive m a ierial w ith a cla m p to h o ld th e s a m p lin g bottle
(figure 1).
5 .3
S ub su rfa ce sa m p ler o f th e type sh o w n in fig u re 2, o r sim ilar, co m p le te with
p la stic ro p e a nd weight.
5.4
T h erm o isolate d p la stic b o xe s w ith c o o lin g p a d s o r sim ila r co o lin g units
(cam ping equipm ent) fo r storag o o f sam ples.
5.5
T h erm o m e te r, 0 to 50° C . p re cisio n - t ° C, p re fe ra b ly unb re a kab le p la stic type,
to be used fo r ch e ckin g tem pe ra ture in p la s tic b o xe s (5>4).
5 .6
Filtration se p a ra tu s fo r 4 .7 cm d ia m e te r m e m b ra n e filters (MF) co nsisting o f at
least three filter fun n els fo r sim u lta ne o us filtra tio n , m a d a o f b orosilicate glass
o r o th e r n o n -to x ic sterilizablo m aterial, c o m p le te w ith e lectric o r w ater va cuum
pum p.
5.7
W ater in cu b a to r fo r 44.5 = 0 .2 ° C.
5.Ô
S te re o m icro sco p e , m a g nifica tio n 10-50X; a n d /o r darkfield c o lo n y counter,
m a g n ifica tio n 2-3X.
5.2
A utoclave, m a x 2 atm ., e lectric o r gas.
5.10
O rvino oven in r srnnii7Atinn a» ip rw n
3
1 ) S u b m e rg e m c u th 2 0 - 2 0 c m
2 ) T u r n e x t e n s io n a rm i a o °
Figure 1.
S ubsurface sa m p lin g w ith e xte n sio n arm.
W ire
M essenger
—
G la s s t u b e (b re a k a b le )
RubDer nose
S te r ile s a r n o Ic b o t t le
H e a v y b o t t o m w e ig h t
Figure 2.
S am pler fo r sterile su bsu rfa ce sa m p lin g.
4
5.12
S tainless steel forceps.
5.13
A na lytica l balance, precision ± 1 mg.
5.14
R efrigerator 4 ; t ° C.
5.1 5
V ib ra to r (shaker} fo r m ixin g liq u id s in cu lture tu b e s.
5.1 6
Petri dish es o f b oro siiica te glass, d ia m ete r 5 cm , co m p le te w ith stainless steel
co nta in ers fo r sterilization, or d isp o sa b le pre-sterilized plastic Petri dishes.
5.1 7
E h rle n m e y e rfla s k s o f b oro siiica te g la ss fo r m e d ia p re pa ra tio n, capacity 1 and
2 litres.
5.1 3
B orosicila te glass b acteriological cu lture tubes.
5.19
T otal v o lu m e (blow -out) boros¡ct:aíe g la ss p ipettes o f 1 , 9 , 10 a nd 2 0 ml
ca pa city, w ith stainless steel co nta in ers fo r sterilization.
N o te : 9 m l ca pa city p ipettes are useful, b u t n o t essential.
5-20
G raduated b cro silica te cla ss cylin d ers o f 100, 500 a nd 1CCC m l capacity w ilh
glass beakers for cover.
5.21
S m a ll b oro siiica te g ia ss tu o e s 6 x 5 0 m m ("D urham vials"! to b e in se rte d in
cu lture tubes (5.18).
5.22
B acte rio lo g ical lo o p s m ade fro m 2 2 «24 C hrom e! g a u g e , n ich ro m e o r platinum«
iridium . D iam eter o f th e lo o p : 3 m m
5.23
H e a vy w ra p p in g paper.
5.2 4
A lu m in iu m foil (h o use h old q u a lity ).
5.25
M e m brane filters (MF), p ore size 0.45}/, d ia m ete r 4.7 cm , or sim ilar, fitting
filtration a pparatus (5.61, c r m e m b ra n e filter o f s im ila r diam eter fitting the
filtration apparatus.
N o te :
The 0.45p p ore size m e m b ra n e filter (MF) s h o u ld be certified b y the
m a n ufa cture r to be free fro m su bsta n ce s w hich m ay hinder th e grow th
a nd d eve lo p m en t of bacteria. M a xim u m re co veries are o b ta in e d using
m em branes co m p o s e d o f m ix e c esters o f cellulose.
5.26
M etal b o xe s w hich aro w a tertigh t a n d suitable lo r in cu b a tin g Petri dishes in the
w ater b ath (5.7).
5.2 7
Filter paper.
5.28
W ater p ath 44.5 .= 0 .2 ° C.
5
B.
C U L T U R E M E D IA , C H E M IC A L S A N D S T O C K C U L T U R E
N o te :
$ .1
“ he co m p ositio n of th e m e d ia rs b a se d o n o ne litre so lu tion s o r sim ilar
units. Before preparation, th e actual n e e d s have to be established and
adequate a m o u n ts m ust b e chose n a ccordin g ly.
M -F C A g a r
6 .1 .1
M e d iu m
T ryp ton e
P roteose p e p to n e No. 3
Y east e xtract
S o d iu m ch lo rid e
L actose
Bile salt N o. 3
A nilin e blue
Agar
D istilled w a te r ($.7)
tO.O
5 .0
3 .0
5 .0
12.5
1.5
0.1
15.0
1.0
g
g
g
g
g
g
g
g
litre
P reparation: Dissolve th e c o m p o n e n ts o f th e m e d iu m in 1 litre o f distilled w ater
{6.7). H eat to boiJmg p o in t until all co m p o n e n ts are co m p le te ly d issolved. A d d 10 m l
o f a 1 p e r cent so lu tio n o f ro sa lie a cid (6. 1.2) keep b o ilin g fo r m o re tha n o n e m inute,
then c o o l. The final m edium sh o u ld have a pH o f 7.4 ± 0 . 1 ; a djust pH , if necessary,
w ith dilu ted a nalytical g rade HCI. C o o l Jo a b o u t 45° C a nd p o u r 4-5 m l into each Petri
dish. A fte r th e a ga r nas solidified in tn e P etri dishes, invert th e Petri dishes a nd store
them in th e refrigerator. The dishes w ith p re pa re d m e d iu m can be k e p t in a refrigerator
for 7 days.
N o te :
T h e a d d itio n o f rosalie a cid is facultative a n d sh o u ld only d e a d d e d , 11
necessary, to suppress excessive g ro w tn o f nom faeca! coliform s.
N o te :
D o n o t autoclavo th e m e dium .
N o te :
The a ga r surface should n e t b e co m e to o d ry b e ca u se if the a ga r is dry
th e MF will n o t adhere w ell to the a ga r su rface (8.5) a nd hinder the
d iffu sio n o f nutrients to th e co lo nie s o n the MF.
6 .1 .2
R o s a lie a c id
Prepare a sufficient a m o un t of 1% solution o f rosalie a cid in 0 .2 N NaOH.
N o te :
6 .2
S olutio n o f rosalie a c ic sh o u ld be p re pa re d freshly each tim e.
s h o u ld n o t be autociaved.
M a c C o n k e y B ro th
6 .2 .1
M e d iu m
S o d iu m faurochofato
5.0 g
i f i n n
It
Ö
NaCi
P ep tcn e
D istilled w a te r <6.7)
5 .0 Q
20.0 g
i 0 lime
P reparation: Dissolve co m p o n e n ts b y sh a k in g . A d ju st pH to 7.1 * 0.1 with
dilu ted HOI a n d the n a od the b ro m o *creso l p u rp le so lu tio n (6.2.2). A d d inverted vials
(5.21) to cle a n cu lture tu b e s (5.18, 8.1) a nd d isp en se su fficien t m e d iu m into th e culture
tu b e s so th a t the in ve rted vials are a t least p a rtia lly co ve re d a fte r the e n tra p p e d a ir in
these vials has been driven o u t during autoclavrng a n d clo se th e tu b e s w ith co tto n
plugs. A uto cla ve (5.S) the c lo se d cu lture tu b e s a t 121° C fo r f C m inutes.
6 .2 .2
B r o m o - c r e s o i p u r p le s o lu tio n
P reparation: D issolve 1 g o f b ro m o -cre so l p u rp le in 9 9 m l o f 95% ethanol.
6 .3
B r illia n t G re e n B ile B ro th
O xgall, d e h yd ra te d
Lactose
P eptone
B rillia nt green
D istilled w ater (6.7)
2 0.0
10.0
10.0
T3.3
1.0
g
g
g
mg
litre
P reparation: D issolve co m p o n e n ts b y sh a kin g . A d d inverted vials (5.21) to
clean c u ltu re tu b e s (5 .1 8 .6 .1 ) a nd dispense su fficien t m e d iu m in to th e cu iture tu b e s so
tna t th e in ve rte d vials are at ¡east partially co ve re d a fte r the e ntrapped a ir in th e se vials
has b e e n driven c u t during autociaving a n c clo se th e tu b e s w ith c o tto n p lu gs. Sterilize
b y autoclavrng (5.9) a t 1 2 !° C. preferably fo r 12 m in u te s, b u t n o t exceeding 15 m inutes.
A fter sterilization, co e i th e b ro th as qu'Ckfy as p o ssib le . Final pH sh o u ld b o 7 .2 s 0.2.
T e st th e sa m p le s o f th e finished p ro d u c t fo r p e rfo rm a n ce using co ntro l s lo c k cultures
(
6 . 10) .
6 .4
I n d o le T e s t S o lu tio n s
6 .4 .1
T r y p to n e w a te r
Try p io n e
N aC i
D istilled w ater (6.7)
10.0 g
5 .0 g
i .0 litre
Preparation- D issolve in g red ie n ts in d is tille d w a te r (6.7). D ispense 5 m l into
e ach te s t tub e (5.18) a nd auto cla ve (5.9) a t 121e C fo r 15 m inutes. The pH sh o u ld be
7.C - 7.4. i‘ necessary a d ju s t w ith dilu ted NaO H b e fo re sterilization.
6 .4 .2
K c v a c 's in d o le re a g e n t
P aea d im e to yl -am w xi-oanzaldehy d e
A m yl a lcohol
C o n ce n tra te d h y d ro ch lo ric acid, HCl
5 .0 g
75.0 m i
25.0 m l
7
P reparation: Dissolve th e bon za id en yd o in am yl a lcohol a nd a dd h ydro ch lo ric
acid. The reagent sh o u ld be yellow.
6 .5
P h o s p h a te B u ffe r (pH = 7.25
KgHPO^
KH2P 0 4
D istilled w a te r (6.7)
6 .5 .1
3.0 g
1.0 g
1.0 litre
P - b u ffe r f o r filt r a t io n
P reparation: D issolve co m p o n e n ts a n a auto cla ve (5 5 ) a t 121° C fo r 15 m in.
6 .5 .2
R -b u ffe r f o r d ilu tio n s
P reparation; Dissolve co m p o n e n ts a n d dispense 9 m l In te s t tu b e s used for
d ilu tion s in th e d ilu tio n teclee {8 <x] a nd auto cla ve (5.9) a t 121° C to r 15 m ín.
6 .6
T h io s u lp h a te S o lu tio n
10 p e r c e n t sodium thiosulphate so lu tio n in d istille d w a te r (6.7) a nd sterilize by
tiltration \e.g . th ro u g h a steiriize d MF (6.2.4, 8.2.5)).
6 .7
D is tilte d W a te r
Use o n ly w ater distilled in all-gisss o r all-quartz distillation apparatus. Dei o n c e o w ater is aiso a cce p ta b le rf p ro c u c e d in a p p a ra tu s n o t releasing to xic
substances.
N o te : C o m m e rc ia l!/ available cisM ied w ater is often p ro d u c e d in c o p p e r a nd
zinc a pparatus a nd is hig hly to xic to r co lifo rm s. B efore u sin g su ch w a te r Its toxicity
sn o u ld be ch e cke d w ith a s to c k cu lture o f £ c o li (6 . 10).
6 .8
D e te r g e n te fo r C le a n in g G la s s w a re a n d A p p a r a tu s
Use o n ly detergents re co m m e n d e d b y th e su p p lie r for b acte rio lo g ica l use. If
such a detergen t is n o t available, ch eck n o rm a l h o u s e h o ld detergents w ith a biotest
using a s to c k cu lture o f £ c o li (6.105.
N o te : Never u se to x ic c h ro m ic -s u lp h u ric acid m ixtu re fo r cleaning glassware.
6 .9
9 5 p e r c e n t E th a n o l p e r
6 .1 0
S t o c k C u ltu r e o f E. c o li.
7.
S A M P L IN G
A n a ly s is .
Details o f a s a m p lin g pían are p ro vid e d in Part I o f th e se guidelines.
8
7 .1
S a m p lin g o f S u r fa c e W a te r
A ttach clean sterilized sam p le b o ttle (8.2.1) to the cle a n sa m p lin g ro d (5.2).
im m e d ia te ly before su b m e rg in g the s a m p le bottle, re m o ve the g ro u n d g la ss stop p er
fro m th e bottle w ith o u t to u ch in g th e s to p p e r eone. Im m e rse th e bottfe fro m th e b o w of
th e b o a t o r fro m th e w in d w a rd side w hile th e b o a t is m o vin g forw ard slow ly. Push the
b o ttle w ith th e s a m p lin g ro d 25 c m u n d e r th e w ater surface w ith th e m o u th o f th e bottle
d o w nw a rd s, in o rd e r to a vo id co n ta m in a tio n o y su rface film , the n tu rn th e sam p le b o ttle
u p w a rd s a nd ta k e th e sam p le (figure 1). T h e sterilize d s a m p le b o ttle m ay a lso be filled
directly b y hand.
Retrieve th e b o ttle a nd disca rd so m e w ater, if necessary, so tha t s o m e air
sp ace re m a in s In th e d o s e d b o ttle . This sp ace is n e e d ed fo r h o m o g e n izin g the w ater
sam p le at th e receiving laboratory. R eplace th e g la ss s to p p e r a n d store th e sam ples
in th e cle a n th e rm o iso la te d box (5.4) w ith c o o lin g p a d s at a b o u t 4° C . Keep sam ples
in th e d a rk a vo id in g exposure to m o re th a n + 1 0 ° C . S eparate b o ttle s fro m each o th e r
w ith clean w ra p p in g p a p e r (5.23) to a vo id b reakage. C h e ck th e tem pe ra ture w ith a
th e rm o m e te r (5.5) every three hours. R e p ort irregularities in th e te s t report. Lábef
sam p le b ottle s in d icatin g the sa m p lin g sta tio n , tim e o f s a m p lin g a n d o th e r facto rs
relevant to th e in terpreta tion o f th e results.
7 .2
S a m p lin g o f S u b s u r fa c e W a te r
L o w e r th e sterilized su bsu rfa ce s a m p le r (8.2.2) after a tta ch in g it to a clean
plastic ro p e, w ith o u t lotting th e w e ig h t d isturb th e b o tto m se dim en ts (figure 2). Release
the m e sse n g e r a nd a fte r o ne m in u te retrieve th e sa m p le r a nd store it in a
th e rm o iso la te d b o x (5.4). P roceed as fo r s a m p lin g o f surface w ater (7.1).
N o te :
it is know n th a t the die-aw ay rate o f co lifo rm s at a m b ie n t tem perature
in th e presence o f light is very h ig h . T herefore, all efforts sh o u ld be
m a d e so as n o t to c o lle c t m o re sa m p le s tha n can b e filtered and
in cu ba ted the sa m e day. If this is n o t p o ssib le , the sam ples sh ou ld
be stored a t 4 ° C and analyzed n o t la ter th a n 24 hours after sa m p lin g.
The w a te r sam p le represents th e test so lu tio n .
8.
TEST PROCEDURE
8 .1
W a s h in g o f G la s s w a re a n d E q u ip m e n t
All g lassw are a nd a pparatus (5) sh o u ld b e w a s h e d w jth non-toxic detergent
(5.8), firs t rinsed th o ro u g h ly w ith h o t tap w a te r a nd the n rinsed a t least three tim e s w ith
d istille d w a te r (S.7).
8 .2
S t e r iliz a t io n o f G ta s s w a re a n d E q u ip m e n t
8 .2 .1
S urface sam p le b ottle s (5.1). C le a n the sam p le b o ttle as described
u nder 8 .1 . Dry a nd sterilize it in a drying oven (5.10) fo r three hours at 160° C. Before
sterilization, p la ce a sm a ll p ie ce o f filter p a p e r (5.27) in th e neck o f th e b o ttle to prevent
the g ro u n d g la ss s to p p e r fro m stickin g a fte r c o o lin g . A fter co o lin g to a m b ien t
9
a nd fit th e g ro und g la ss stop p er securely into the n e ck o f th e b o ttle . P ut the b ottle s into
detergent-cleaned the rm oisola te d b o xe s (5,4). S eoaratc the b ottle s from each other
w ith clean w ra p p in g p aper (5.23) to a vo id breakage.
N o te :
if re sidual chlorine is su sp e cte d in tn e w a te r sa m p le , add, aseptlcally,
O.t m l o t a tO per c e n t th io su lp h a te c c tu tlo n (6.6) fo r e a ch 100 m l
s a m p le to th e contents o f th e sam p le b o ttia b e fo re sterilization. This
a m o u n t j $ sufficient to neutralize a b o u t 15 m g o f residual ch lo rin e per
litre.
8 .2 .2
S ubsurface sa m p le r (5.3). Clean th e su bsu rfa ce sa m p le r as described
u nder (8.1), rinse w ith ta p a nd d istille d w ater (6.7). E nclose each sa m p le r in heavy
w ra p p in g p aper (5.23) or alum inium foil (5.24) a nd sterilize in a n autoclave (5.9) fo r 15
m inutes a t 121° C.
8 .2 .3
Petri dishes (5.15) a nd p ip e tte s (5.19). Clean dish es and pipettes,
co m p le te w ith a c o tto n plug in th e m o u th p 'e ce , are p u t into suitable stainless steel
containers a nd sterilized in a dfym g even (5.10) fo r three hours a t 160° C.
N o te :
D isposable pre*stenlized p la stic Petri dish es m ay be m o re e conom ical
to u se than reusable g la ss Petri dishes.
8 .2 .4
Filter funnels o f filtration a pparatus (5.6). Loo se n the filter-holding
assem bly slig h tly a nd w ra p th e w h o le filter funnel in heavy w ra p p in g p aper (5.23) or
a lu m in iu m foil (5.24), Sterilize in an autoclave (5.9) fo r 15 m in u te s at 121° C, o r in a
drying oven (5.1C) fo r 3 hours at 16C° C.
8 .2 .5
M em brane Filters (MF) <5.25). R em ove the p aper se pa ra tor (if present)
a nd p la ce 10 to 12 clean MFs into Petri dish es (5.16). A utoclave <5.9) for 15 m jnutes
at 121° C. A t the e nd o f the sterilization tot the steam escap e ra p idly in o rd e r to
m inim ize th e accum ula tio n o f cond e nsa to o n the MFs.
N o te :
S terilized M Fs am co m m e rcia lly available.
8 .2 .6
F o rce ps (5.12).
ethanol (6.9) and fla m in g thorn.
8 .3
Stenii?© forcep s by dip pin g th e m into 95 per cent
S e le c tio n o f S a m p le S í¿ c a n d D ilu tio n S e rie s
The MFs sn ou ld ideally have Ir o n 20 lo 8 0 co lo nie s after in cu ba tio n. If
previous experience fo r planning the d ilu tio n senes fo r e'ean seaw ater ;s not available,
filter the follo w in g vo lu m e s o f the o rig in al sa m pie: 100 m:, 10 m l, 1 m i a nd 0.1 mi
(figure 3). For co nta m in a te d w aters the d ilu tion s have to be greater.
8 .4
P re p a ra tio n o f th e D ilu tio n S e rie s
B efore taking aliquots fro m the o ng nal sam ple o r from th e d ilu tio n s these m ust
b e vig o rou sly shake n in o ir.e r to guar antee th a t re p rcscn ta tw e a liq u o ts are taken.
10
T e s t s a m p le
P * b u ffe r
0-0
1
2 0 m l each
D -1
( 3 lim e s )
1 m l*
1 ml
10 m l
100 ml
D 2
1 ml
t ml
7
0 .0 1 m i
0.1 m i
1 ml
10 m l
100 ml
r.f o r i g i n a l s a m p le o n M F
Figure 3.
P reparation of d ilution series a n d filtration procedure.
P repare th e dilution series by taking w ith a sterilized p ip ette (8.2.3), after
vig o rou sly sh a kin g th e sam p le (7), I m l iro m th e o rig in al sam p le (figure 3, dilution: 0-0)
and transfer th is 1 m l into a cu lture tube c o n ta in in g 9 m l o f P -buffer (6.5.2) to m ake the
first d ilution (D-1). Agitate th e tub e c n a m ixe r (5.15) o r sh ake it vig o ro u sly b y hand.
C o n tinu e th e preparation o f th e d ilution senes b y ta k in g 1 m l fro m the firs t dilution (DO )
a nd m ixin g il in a n e w culture tub e co n ta in in g 9 m l o í p h o sp ha te b uffer (6.5.2) in o rd er
to o b ta in the se co n d d ilution (0 *2 ), etc.
8 .5
F ilt r a t io n P ro c e d u re
Beqm futratron w ith the hig he st d ilution (e.g. D*2) in o rd er to avoid co nta m in a tion
from sa m p ie s co nta in in g b acte ria in n ig h e r co n ce n tra tio n s. U se a sterilize d filtration
funnel (8.2.4) fo r e a ch d ilu tio n series. Place the sterilize d M F (8.2.5) w ith flam ed
sterilized forcep s (8.2.5) o ver the p o ro u s plate o f th e filtration a p p a ra tu s (3.2.4).
C arefully place th e m atching fu n n e l u nit o v e r th e re ce pta cle a nd lo ck it in place. A dd
into th e funnel a b o u t 2 0 m l o f su ffe r so lu tio n (6.5.1). W ith a sterilized p ip ette (8.2.3) a nd
1 m l o f the D-2 d ilu tio n into th e b uffer s o lu tio n in th e fun n el. F ilter w ith a partial vacuum .
W ash th e funnel walls w ith a pp ro xim a te ly 20 m l o f b u ffe r so lu tio n (6.5 1).
11
Fitter w ith e partial vacuum . W ash th e funnel w alls tw o m o re tim e s w ith 2 0 m ( o f buffer
s o lu tio n Í6 .5 .1). U n lo c k and rem ove th e funnel, im m ed ia tely rem ove the M F w ith flam ed
steniized forcep s (6.2.6) and place the M F c n th e agar su rface o f th e m e ciu m co nta in ed
in the Petrj dish (6.1.7) with a ro lling m o tio n to a v c id th e e ntra p m e n t o f air. Before
filtering tn e n e x t d ilu tio n (D-1) in th e sam e m anner, pass 20 m l o f buffer solution (6.5.7)
th ro u g h th e a sse m b le d filtration unit.
8 .6
I n c u b a tio n
The Petri d ish e s co nta in in g th e M Fs on a ga r (8.5) are sealed a nd im m ediately
p la ced h orizo n ta lly in sid e clean m etai boxes. These m e ta l b o xe s are the n p la c e d in a
w ater b ath (5.7) a n d in cu ba tea im m ediately fo r 24 h o u rs at 44.5 ± 0.2° C. A s a sterility
cn eck, in cu b a te a lso o ne b la n k {w ithout MF), i.e. a Petri d ish co n ta in in g a ga r (6.1.1)
only.
N o te :
8 .7
Meta) b o xe s m u st be suitably w e ig hte d to prevent them fro m floating.
in te r p r e ta tio n
C o u nt w ith a sioreom tcroscope o r sim ila r (5.8) o n ly co lo nie s w h ich a p p e a r as
blue co lo u re d . If the num ber o f d ubious co lo nie s is greater tha n 10 p e r cent o f the total
n u m b e r o f c o lo n ie s, te s t d ubious co lo nie s either b y M a cC o nke y b ro th te s t (8.9.1) or
brilliant g reen bile b ro th test (8.9.2).
in areas w h e re industries discharge p o fysa ccha rite s (paper m ills, sugar best
industries, etc.), fu rth e r co nfirm ation b y the in d ole te s t m ay b e necessary (8.9.3).
N o te :
8 .8
C o lo nies p ro d u ce d b y faecal co fifo rm b acte ria are blue in co lo ur. The
non*1aoca) coi fo rm co lo nie s are grey to ere am -cotoured. B a ckg ro u nd
c o lo u rs o n the m e m brane filter will vary fro m a yellow ish cream to a
faint blue, d e p e nd in g on th e age c f th e ro sa lie acid salt re a g e n t
N orm ally, onry few norvfaecal conform co lo nie s will be observed o n mFC a ga r because o í the selective a ction o f th e elevated tem perature
a nd a ddition o f the rosalie a cid salt reagent,
E s tim a tio n o f P re c is io n
C h o ck th e p re cisio n c f th e te ch n iq ue a t p e rio d ic intervals (at least o n ce every
season) b y pre pa rin g three in d ep e nd e nt series o f d ilu tion s (8.4) u sin g the sam e sam ple,
i.e. re peating the fo u r last co nse cu tive e lu tio n steps d e scribe d u nder 8.3 a nd 8.,4
(figure 3). The w ater sam p le used sh ou ld be c o lle cte d d u rin g th e routine m o n itoring
p ro g ra m m e a t a c o a s ta l stag o n typica) p i th e area. T h e d ilu tio n series sh o u ld b e
selected in such a w a y so tha t o ne d ilution step yields three M F co un ts w hich satisfy
the 20 to 2 00 co lo nie s requirem ents expressed in se ctio n 9,1.
Filter each in d ividual d ilution Jollow.ng p ro ce d u re 8,5. incubata a c co rd in g to
p rocedure 6.6. R eport M F co un ts fo llo w in g th e p ro c e d u re d e scrib e d m se ction s 9.1 and
9.2 taking into co nside ra tion interpretation m e tho d o f section 8.7. Results sh ou ld be
reported in th e te s t re p o rt (table 2, item 9),
12
C a lculate th e faecal conform c o n ce n tra tio n s o f th e o rig in al sam p le fo r each o f
the re p lica te results, a ccordin g to section 9 .3 a nd re p o rt th e results in th e te s t report
(lao ie 2 , ite m 10).
For each d ilution step having th e three M F co u n ts betw een 20 a n d 2 00 faecal
c o lifo rm c o lo n ie s, calculate: the m ean co n c e n tra tio n , th e co n ce n tra tio n range, th e
sta n d a rd d eviatio n o f the co nce n tra tio n s, a nd th e co e fficie n t o f va ria tio n o f th e
co nce n tra tio n s, a nd re co rd th e m in the te s t re p o rt (table 2 , item 11).
If th e s a m p le does n o t y e ld a t le a st 2 0 co lo n ie s per m e m brane filter in o ne
d ilu tio n , p re p a re a te s t so lu tio n fro m a s to c k cu lture (6.10) a n d repeat th e estim ation of
precision.
NOTE:
8 .9
C oefficient o f variation (%) » stan d ard deviation x JQQ
m ean
C o n firm a to ry Test
8 .9 .1
M a cC o nke y b ro th test: W ith a fla m e d b a cte rio lo g ica l lo o p (5.22)
tra n sfe r th e su sp e cte d c o lo n y fro m m e M F in to a c u ltu re tub e co n ta in in g M acC onkey
b ro th (6.2.1) a nd incubate (5.28) at ¿4,5 ± 0 .2 ü C fo r 24 h o u rs. C o lifo rm s wilt d evelop
gas w h ic h is tra p p e d in the inverted D u rh am tu b e s, a nd a cid w h ich turns the vioieW ike
c o lo u r o f th e o rig in a l broth into a yellow ish co lo ur.
8 .9 .2
B rillia nt green bile b ro tn te s t: W ith a flam ed b acte rio lo g ica l lo o p (5.22)
transfer th e su sp e cte d c o lo n y fro m the M F into a cu lture tub e co nta in in g b rilliant green
bile b ro th (6.3) a nd incubate (5.28) at 44 5 ± 0 .2 ° C fo r 24 hours. C o lifo rm s w ill develop
gas w h ic h is tra p p e d in the inverted D u rh am vials.
N o te :
The M acC onkey b ro th te s t is e qu iva le nt to th e b rilliant green bile b ro th
test. Either can bo used fo r co nfirm ation.
8 .9 .3
in d o le test: W ith a fla m e d b a cte rio lo g ica l lo o p (5.22) transfer th e
su spe cted c o lo n y into culture tu b e s co n ta in in g try p to n e w ater (6.4.1) a n d incubate at
44,5 ± 0 .2 " C in a w ater b ath (5.23) fa r 24 hours. T h en a d d C.2 - 0.3 m l of K ovac’s
in d ole re a g e n t (6.4.2) a nd sh ake . Let th e tu b e sta n d fo r a b o u t 10 m in u te s and observe
th e results.
A d ark ro c c o lo u r in t ne am yl a ico n o l surface la ye r co n stitu te s a p ositive indole
tost; th e o rig in al c o lo u r of th e reagent, a negativo test. A n o ra n g e co lo u r p ro b a b ly
in d icate s th e p re s e n c e o f skatoie a nd m ay b e re p o rte d as a p ositive reaction.
9.
E X P R E S S IO N O F R E S U L T S
9.1
R eport Ih c n u m b e r o f co lifo rm c o lo n ie s o n in d ivid u a l M Fs after the in cu ba tio n
has been co m p le te d a nd a djust this c o u n t after th e co n firm a to ry te sts, if necessary,
have been m a c e . U se only M Fs w ith a total n u m b e r o f co lo n ie s (i.e. co lifo rm s p lu s non*
13
coliform s) betw een 20 a nd 200. Retain only îw o sig n ifica n t d ig its o f th e co un ted
n u m b e r o f co lifo rm colonies per filter
Indicate th e results o btained fo r each filter se pa ra tely in ih e test re p ort (table
1, item 9).
9 .2
E xpress the results m term s o f faecal conform s p e r 100 m l o f sa m ple, using the
follow ing equation:
faecal ca lifo rnia p e r 100 m l sam p ie = a d ju ste d n u m b e r o f co lifo rm co lo nie s x 10Q
m i o f sam p ie filtered
in d ica te the results o b ta in e d fo r each d ilu to n separately in th e te s t re p ort (table
T, item 10). R eport aiso th e results o b ta in e d o n M Fs w ith loss tha n 2 0 co lifo rm colonies
per filter, if tho re aro no co liform c o io d e s o n th e filter re p o rt th e re su lts as " < 1 co liform
per 100 ml".
9 .3
C o m p u te th e n u m b e ro f faecal c o lifo rm s p e r 100 m l sam p le a nd re p o rt It a s the
final te s t result (table 1, item 11), if the re are MFs c o n ta in in g betw een 20 a nd 200
characteristic co lo nie s in tw o co nse cu tive d.lutions c a lcu la te th e m e a n o f th e se dilutions
and re p o rt it as final te s t result.
9 .4
R ecord in the test re p ort (table 1. item 12) a no m a lie s observed in lest
p ro ce d u re (confluent g ro w th o f colonies, deviation fro m tem pe ra ture p re scrib ed for
sam ple storag e and incubation, etc.).
10-
TE S T REPORT
Table 1. Faecal co lifo rm s in seaw ater sam ples.
1 S a m p lin g a re a
1
2. S a m p lin g p o in t
c o u n tr y :
a re a :
_
c o d e n u m b e r;
(S ta tio n )
lo n g it u d e :
la titu d e :
3 . T im e o t s a m p lin g
day:
h o u r:
m o n th :
4 S a m p lin g a n d e n v iro n m e n t c o n d itio n s
S a m p 'in g d e p th :
vean
C o n ta in e r n u m b e r:
T e m p é ra tu r e a t s a m p lin g c e p th :
S a lin ity a t s a m p lin g d e p th :
D u ra tio n o t s to ra g e :
(o th e r fa c to r s w h ic h m a y in flu e n c e th e r e s u lts s h o u ld b e r e p o n e d u n d e r 12 }
5 T im e o f filtra tio n
5 . S ta n o f in c u b a tio n
h o u r:
7. E n d o f in c u b a tio n
hout
8 . C o n firm a to ry te st
h o u r:
M acC onkev:
d a y:
/ ___ /
day:
d a y : ___ / ___ / ____
O ni lia n t g re e n .
In d o le te s t:
9 N u m b e r o f c o lo n ie s p e r in d iv id u a l filte r
m l o f o rig in a l
D ilu tio n
D-0
0 -0
D-0
D-1
12.
12
s a m p le filte re d
¡ ____¡
t o . C o lo n ie s o f F. c o h fo r m s /1 0 0 m l
F a e c a l c o lifo 'm s
D ilu tio n s
« o f./100 m i
c o lo n ie s
100
to
i
D-2
3.1
0.01
D-3
D-4
0-031
0.C001
11. T e s t re s u lt
F . c o lifo rm is /1 0 0 m í
A n o m a lie s o b s e rv e d in th e te s t p ro c e d u re
F u ll a d d re s s o f th « in s titu tio n w in c h
c a rr ie d o u t th e a n a ly s is :
1i
N a m e ts ) a n d s ig n a tu r e s ) o t th e
p a ra o n is ) w h o e a rn e d o u t th e a n a ly s is
Table 2.
1.
P recisian sstim a iio n for faecal co lifo rm s determ ination.
S a m p lin g a r c a
c o u n tr y
a re a ;
2 S a m p lin g p o n i'
(s ta tio n )
3 . T im e o f s a m p lin g
h o u r:
code num ber
lo n g itu d e :
la titu d e :
d a yt
m o n th :
ve a h
4. S a m p lin g a n d e n v iro n m e n t c o n d itio n s
S a m p lin g d e p th :
T e m p e ra tu re a t s a m p lin g d e p th *
C o n ta in e r n u m b e r.
S a lin ity a r s a m p lin g d e p th :
D u ra tio n o f s ro ra o e :
( o th e r fa c to rs w hsch m a y in flu e n c e th e re s u lts s h o u ld D e re p o r te d u n d e r 12)
5 . T im e o f fitr a tlo n
h o u r.
h o u r:
6 , S ta rr o f in c u b a tio n
7 £ n d o f in c u b a tio n
9. C o n firm a to ry te st
d a y ; ___ / ___ / ___
day:
/ __/
n o u i:
< * a y :___ / ___ / ___
B rillia n t p re e n :
In d a le ts s c
M acC onkey
9 . N u m b e r c f c o lo n ie s p e r .n o iv io u a . filte r
D ilu tio n
m l o f o r ig in a l
s a m p le filte re d
10. C o lo n ie s o f F c a ( i» o r m s /l0 0 m l
F . c o lifo rm s c o lo n ie s
re o u ca
ts t
2nd
D ilu tio n s
C O I./1 00 m l
3 rd
11. R e s u lts (F. c o h fo r m s /1 0 0 m l)
----------------
12.
13,
m ean.
ra n a « :
s t d .d e v :
c o e f.v a r.r
--------------------- ------------------------------------— — •
%
A n o m a lie s o b s e rv e d rn th e te s t p ro c e d u re
F u ll a d d re s s o t th e in s titu tio n w m cn
e a rn e d c u t th e a n a ly s is :
14
N a m e {s ) a rid s ig n a tu r e s ) o f th e
p e rs o n {$ ) w h o e a rn e d o u t th e a n a ly s is '
te
11.
REFERENCES
APHA (1981) S ta n d a rd m e thods fo r th e e xam ina tio n o f w ater and w a ste water.
A m e rica n P ublic HeaJth A ssociatio n , W a sh in gto n D.C. (15th edition).
U N E P /W H O (1985) D eterm ination o f faecal co lifo rm s in seaw ater b y th e m ultiple-tube
(MPN) test. Reference M e thods fo r M arine P ollution S tudies, N o. 22 UNEP,
Geneva.
J N E P /W H O (1988) G uidelines fo r m o n ito rin g th e q ua lity o f coastal recreational and
sh e llfish -cro w in g waters. R eference M e th o d s fo r M arine P ollution S tud ie s No.
1, UNEP, Geneva.
W H O /U N E P (1983) M e thods for m o n ito rin g se le cte d p o llu ta n ts in sew age effluents and
coastal recreational w ater R eport on a W H O /U N E P jo in t m e eting. Rom o 24-26
N ovem ber 1982. D o cu m en t J C P /R C E 2 1 1(2), W H O re g ion a l o ffice for Europe,
C openhagen.
Issued bv
Occa 11$ a n d Coastal A rcas P rogram m e A c tiv ity Centre
U n ite d N a tio n s E n v iro n m e n t Program m e
A d d itio n a l copies o f tlu s p u b lic a tio n
can 1« olxanted from .
Oceans a n d Coasta) A reas P rogram m e A c tiv ity Centre
U n ite d N ations E n v iro n m e n t Program m e
P.O . H o x « » .S /
Nairobi
KENYA
o r fio tn :
M a rin e E n v iro n m e n ta l Studies la b o ra to ry
In te rn a tio n a l A to m ic E n e rg y Agency
M a m ie E n v iro iiitic n t La b o ratory
B .P . N o. 8 0 0 * M C y 8012
M O N AC O CEDEX